Stroke remains the third leading cause of death in the US; yet attempts to devise therapeutic interventions have largely been frustrated. Recently neural stem cells have been implicated in processes that lead to neuronal regeneration following stroke, raising the possibility that it might be feasible to ameliorate brain injury by boosting neuronal regeneration. Our recent evidence suggests that neurogenesis following ischemic brain injury might not only lead to the replacement of damaged cells but also contribute to functional recovery, thereby further supporting the notion that strategies which, augment endogenous neurogenesis may hold clues for the development of stroke therapy. Our long-term goal is to implement a therapeutic strategy based on neuronal regeneration. Central to neurogenesis events at the molecular level are a group of membrane receptors that include the stem cell receptor c-kit, the erythropoietin receptor EpoR and the insulin-like growth factor receptor IGF-1R. Despite a wealth of literature on c-Kit/EpoR/IGF-1R, astonishingly little is known about their molecular environments in the membrane and whether a direct interaction underlies an intriguing functional synergism reported for these proteins. Membrane protein assemblies are notoriously difficult to study; most approaches require them to be solubilized by detergents despite their unpredictable tolerance to such treatment. We have developed a novel method that covalently preserves protein interactions through time controlled transcardiac perfusion cross linking (tcTPC) prior to the disruption of tissues integrity to capture these proteins in the context of their physiologic milieu. The objective of this proposal is to determine the molecular environment of ckit/ EpoR/IGF-1R in normal condition and after cerebral ischemia. To achieve this, we will first carry out a tcTPC based comparative interactome analysis of the c-Kit/EpoR/IGF-1R environments in brain and peripheral organs that will reveal key interactors of these receptors. We will then look for dynamic regulators of c-Kit/EpoR/IGF-1R by comparing brains of experimentally stroked and control animals. The cross linked regulators of interest will be co-purified by high stringency affinity-purification using antibodies to c-Kit, EpoR and IGF-1R. Subsequently, immunopurified cross linked complexes will be subjected to proteolytic digestion, two-dimensional (2D) separation of peptides by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). A computationally aided search of genomic databases with mass lists obtained from collision induced dissociation (CID) spectra will reveal the identity of co-purified components and regulators of the c-kit/EpoR/IGF-1 R signaling interactome. To determine the role of candidate interactors we will manipulate their expression levels in cultured neural stem cells by RNAi knockdown and overexpression and will assay for signs of receptor activation by Western blotting analysis and/or phosphorylation of c-kit/EpoR/IGF-1 R following external stimulation with ischemic brain extracts. We anticipate that the identification of proteins interacting with ckit/ EpoR/IGF-1R signaling in the brain in the context of cerebral ischemia will not only contribute to our understanding of endogenous neurogenesis but also assist the development of cell-replacement therapy for treating stroke.